Intratumor heterogeneity has been demonstrated in a substantial number of animal and human tumors. Our laboratory has previously isolated a set of subpopulations from a single BALB/cfC3H mouse mammary tumor, which differ in morphology, karyotype, growth properties in vivo and in vitro, and sensitivity to therapeutic agents. We have shown that certain of these subpopulations can interact to affect each other's growth and sensitivity to chemotherapy, and thus that the properties of the heterogeneous tumor are not simply the sum of the properties of the individual subpoputations. I have demonstrated the inhibition of growth of one subpopulation by a diffusable factor produced by the second subpopulation. I have described interactions between subpopulations which increased the sensitivity of one subpopulation to cyclophosphamide in vivo, and which increased the sensitivity of a second subpopulation to methotrexate in vitro. I present here preliminary data indicating a third such interaction in which the sensitivity of a subpopulation to thioguanine was increased by contact with a second subpopulation. I propose to investigate the biochemical and physiological mechanisms which underly the interactions described here. In order to broaden the number of interactions which can be investigated, and in order to examine interactions between neoplastic and preneoplastic cells as well, I propose to isolate new subpopulations from spontaneously arising BALB/cfC3H tumors and from tumors arising from transplated hyperplastic alveolar nodules. These subpopulations will be characterized as to growth properties and growth interactions using the techniques already developed. The mechanisms for these interactions will also be investigated. For populations which interact in a manner affecting their growth, the effect of this interaction on the sensitivity to the cell-cycle dependent drugs methotrexate, fluorouracil, and thioguanine, will be investigated.